Immature Tissue Research Articles

MUCOGRAFT: 3D COLAGEN MATRIX FOR SOLVING GINGIVAL RECESSIONS

Introduction. Clinical correction of recessions is an important aesthetic and functional moment, but periodontal regeneration is evident only by histological confirmation.Goal. Histologically verify the finding in the intervening segment after applying the mucograft 6 months of treatment. Material and method. A prospective clinical study covered 12 patients transplanted with mucograft. In all subjects after 6 months of intervention, is taken biopsy from a transplanted place with a micropunch gum technique in size of 2 mm, after a previous application of 3% local anesthetic Scandonest. The material is subjected to a histological examination of a tissue sample graded in 4 categories: immature collagen tissue, mature (normal) collagen tissue, fragmented collagen and oedematous tissue. At the same time, the structure of the elastic fibers in the tissue sample material is graded in three categories: with normal structure, fragmented rare and fragmented multiplied elastic fibers.Results. In the majority of the respondents 9 (75%) the presence of mature (normal) collagen fibers was confirmed, while in 3 (25%) immature collagen tissue, I relation to the elastic fiber test, the elastic fibers with the normal structure and elastic fibers with an immature fragmented structure are equally represented.Conclusion. After six months of the intervention, the GeistlichMucograft® 3D collagen matrix is an ideal biomaterial for soft tissue regeneration and is an ideal replacement for the connective tissue graft that is still a gold standard in the resolution of gingival recessions, which simultaneously provides stability for suturing and immeasurable stabilization blood coagulum with early release of soft tissue cells. It is confirmed that the Mucograft collagen matrix histologically is characterized by a shorter epithelium and is successfully incorporated into adjacent adherent host tissues, and at the same time there is a clinical appearance of the natural gingiva of the transplanted site. Mucograft membrane is a promising alternative for the regeneration of keratinized mucous membranes.After six months of the intervention, the GeistlichMucograft® 3D collagen matrix is an ideal biomaterial for soft tissue regeneration and is an ideal replacement for the connective tissue graft that is still a gold standard in the resolution of gingival recessions.

Transcriptome analysis of the curry tree (Bergera koenigii L., Rutaceae) during leaf development

The curry tree (Bergera koenigii L.) is a widely cultivated plant used in South Asian cooking. Next-generation sequencing was used to generate the transcriptome of the curry leaf to detect changes in gene expression during leaf development, such as those genes involved in the production of oils which lend the leaf its characteristic taste, aroma, and medicinal properties. Using abundance estimation (RSEM) and differential expression analysis, genes that were significantly differentially expressed were identified. The transcriptome was annotated with BLASTx using the non-redundant (nr) protein database, and Gene Ontology (GO) terms were assigned based on the top BLAST hit using Blast2GO. Lastly, functional enrichment of the assigned GO terms was analyzed for genes that were significantly differentially expressed. Of the most enriched GO categories, pathways involved in cell wall, membrane, and lignin synthesis were found to be most upregulated in immature leaf tissue, possibly due to the growth and expansion of the leaf tissue. Terpene synthases, which synthesize monoterpenes and sesquiterpenes, which comprise much of the curry essential oil, were found to be significantly upregulated in mature leaf tissue, suggesting that oil production increases later in leaf development. Enzymes involved in pigment production were also significantly upregulated in mature leaves. The findings were based on computational estimates of gene expression from RNA-seq data, and further study is warranted to validate these results using targeted techniques, such as quantitative PCR.

Effect of recombinant human vascular endothelial growth factor on testis tissue xenotransplants from prepubertal boys: a three-case study.

Does recombinant human vascular endothelial growth factor (VEGF-165) improve the efficiency of human immature testis tissue (ITT) xenotransplantation? ITT fragments from three prepubertal boys were cultured for 5 days with VEGF-165 or without (control) before xenotransplantation into the testes of immunodeficient mice. Xenotransplants were recovered at 4 and 9 months post-transplantation and vascularization, seminiferous tubule integrity, number of spermatogonia and germ cell differentiation were evaluated by histology and immunohistochemistry. Transplants from donor 1 and donor 2 treated with VEGF demonstrated higher vascular surface (P = 0.004) and vessel density (P = 0.011) overall and contained more intact seminiferous tubules (P = 0.039) with time, compared with controls. The number of spermatogonia was increased over time (P < 0.001) irrespective of treatment and donor, whereas, for the VEGF-treated transplants, the increase was even higher over time (P = 0.020). At 9 months, spermatocytes were present in the xenotransplants, irrespective of treatment. No transplants could be recovered from donor 3, who had already received treatment with cyclosporine for aplastic anaemia before biopsy. In-vitro pre-treatment of human prepubertal testis tissue with VEGF improved transplant vascularization in two out of three cases, resulting in improved seminiferous tubule integrity and spermatogonial survival during xenotransplantation. Although further studies are warranted, we suggest VEGF to be considered as a factor for improving the efficiency of immature testis tissue transplantation in the future.

Influence of experimental alcoholism on the repair process of bone defects filled with beta-tricalcium phosphate

This study evaluated the effect of ethanol on the repair in calvaria treated with beta-tricalcium phosphate (β-TCP). Forty rats were distributed into 2 groups: Water group (CG, n = 20) and Alcohol Group (AG, n = 20), which received 25% ethanol ad libitum after an adaptation period of 3 weeks. After 90 days of liquid diet, the rats were submitted to a 5.0 mm bilateral craniotomy in the parietal bones; the left parietal was filled with β-TCP (CG-TCP and AG-TCP) and the contralateral only with blood clot (CG-Clot and AG-Clot). The animals were killed after 10, 20, 40 and 60 days. The groups CG-Clot and AG-Clot showed similar pattern of bone formation with a gradual and significant increase in the amount of bone in CG-Clot (22.17 ± 3.18 and 34.81 ± 5.49) in relation to AG-Clot (9.35 ± 5.98 and 21.65 ± 6.70) in periods of 20–40 days, respectively. However, in the other periods there was no statistically significant difference. Alcohol ingestion had a negative influence on bone formation, even with the use of β-TCP, exhibiting slow resorption and replacement by fibrous tissue, with 16% of bone formation within 60 days in AG-TCP, exhibiting immature bone tissue with predominance of disorganized collagen fibers. Defects in CG-TCP showed bone tissue with predominance of lamellar arrangement filling 39% of the original defect. It can be concluded that chronic ethanol consumption impairs the ability to repair bone defects, even with the use of a β-TCP biomaterial.

Complete spermatogenesis in intratesticular testis tissue xenotransplants from immature non-human primate.

STUDY QUESTIONCan full spermatogenesis be achieved after xenotransplantation of prepubertal primate testis tissue to the mouse, in testis or subcutaneously?SUMMARY ANSWERIntratesticular xenotransplantation supported the differentiation of immature germ cells from marmoset (Callithrix jacchus) into spermatids and spermatozoa at 4 and 9 months post-transplantation, while in subcutaneous transplants, spermatogenic arrest was observed at 4 months and none of the transplants survived at 9 months.WHAT IS KNOWN ALREADYAuto-transplantation of cryopreserved immature testis tissue (ITT) could be a potential fertility restoration strategy for patients with complete loss of germ cells due to chemo- and/or radiotherapy at a young age. Before ITT transplantation can be used for clinical application, it is a prerequisite to demonstrate the feasibility of the technique and identify the conditions required for establishing spermatogenesis in primate ITT transplants. Although xenotransplantation of ITT from several species has resulted in complete spermatogenesis, in human and marmoset, ITT has not been successful.STUDY DESIGN, SIZE, DURATIONIn this study, we used marmoset as a pre-clinical animal model. ITT was obtained from two 6-month-old co-twin marmosets. A total of 147 testis tissue pieces (~0.8–1.0 mm3 each) were transplanted into the testicular parenchyma (intratesticular; n = 40) or under the dorsal skin (ectopic; n = 107) of 4-week-old immunodeficient Swiss Nu/Nu mice (n = 20). Each mouse received one single marmoset testis tissue piece in each testis and 4–6 pieces subcutaneously. Xenotransplants were retrieved at 4 and 9 months post-transplantation and evaluations were performed with regards to transplant survival, spermatogonial quantity and germ cell differentiation.PARTICIPANTS/MATERIALS, SETTING, METHODSTransplant survival was histologically evaluated by haematoxylin-periodic acid Schiff (H/PAS) staining. Spermatogonia were identified by MAGE-A4 via immunohistochemistry. Germ cell differentiation was assessed by morphological identification of different germ cell types on H/PAS stained sections. Meiotically active germ cells were identified by BOLL expression. CREM immunohistochemistry was performed to confirm the presence of post-meiotic germ cells and ACROSIN was used to determine the presence of round, elongating and elongated spermatids.MAIN RESULTS AND THE ROLE OF CHANCEFour months post-transplantation, 50% of the intratesticular transplants and 21% of the ectopic transplants were recovered (P = 0.019). The number of spermatogonia per tubule did not show any variation. In 33% of the recovered intratesticular transplants, complete spermatogenesis was established. Overall, 78% of the intratesticular transplants showed post-meiotic differentiation (round spermatids, elongating/elongated spermatids and spermatozoa). However, during the same period, spermatocytes (early meiotic germ cells) were the most advanced germ cell type present in the ectopic transplants. Nine months post-transplantation, 50% of the intratesticular transplants survived, whilst none of the ectopic transplants was recovered (P < 0.0001). Transplants contained more spermatogonia per tubule (P = 0.018) than at 4 months. Complete spermatogenesis was observed in all recovered transplants (100%), indicating a progressive spermatogenic development in intratesticular transplants between the two time-points. Nine months post-transplantation, transplants contained more seminiferous tubules with post-meiotic germ cells (37 vs. 5%; P < 0.001) and fewer tubules without germ cells (2 vs. 8%; P = 0.014) compared to 4 months post-transplantation.LARGE SCALE DATAN/A.LIMITATIONS, REASONS FOR CAUTIONAlthough xenotransplantation of marmoset ITT was successful, it does not fully reflect all aspects of a future clinical setting. Furthermore, due to ethical restrictions, we were not able to prove the functionality of the spermatozoa produced in the marmoset transplants.WIDER IMPLICATIONS OF THE FINDINGSIn this pre-clinical study, we demonstrated that testicular parenchyma provides the required microenvironment for germ cell differentiation and long-term survival of immature marmoset testis tissue, likely due to the favourable temperature regulation, growth factors and hormonal support. These results encourage the design of new experiments on human ITT xenotransplantation and show that intratesticular transplantation is likely to be superior to ectopic transplantation for fertility restoration following gonadotoxic treatment in childhood.STUDY FUNDING/COMPETING INTEREST(S)This project was funded by the ITN Marie Curie Programme ‘Growsperm’ (EU-FP7-PEOPLE-2013-ITN 603568) and the scientific Fund Willy Gepts from the UZ Brussel (ADSI677). D.V.S. is a post-doctoral fellow of the Fonds Wetenschappelijk Onderzoek (FWO; 12M2815N). No conflict of interest is declared.

Abstract B153: Human NK cell distribution memory and residence in tissue sites

Abstract Natural killer (NK) cells are innate immune cells with the ability to kill tumor cells without prior exposure. NK cells express multiple activating and inhibitory receptors in addition to the low-affinity immunoglobulin G binding receptor CD16. Accumulating evidence implicates a role of NK cells in not only direct killing of tumor cells, but also in cancer immunosurveillance and preventing metastasis to tissues sites. However, at present the distribution, diversity and tissue driven differences in NK cell function are not well characterized which may have an implication on the anti-cancer potential of tissue NK cells. Through collaboration with LiveOnNY our local organ procurement organization, we receive blood, bone marrow (BM), lung, intestines, tonsil and associated lymph nodes (LN) from research consented organ donors. Here we used this unique tissue resource to investigate the distribution, phenotypic, functional and transcriptional diversity of NK cell subsets in human tissues. We found that NK cells are ubiquitously distributed in human tissues comprising up to 40% of the CD45+ CD14/19- cells in blood, BM, spleen and lung, while only a small fraction (up to 2%) in the intestine and the LN. While blood, BM, spleen and lung are dominated by mature NK cells (CD56dim CD16+), majority of the NK cells in intestine and LN are immature (CD56hi CD16-). Age, sex and CMV sero-status do not show any correlation with NK cell distribution or subset frequency in tissues. Notably, NK cell subset distribution seems to drive functional differences between tissue NK cells, with lymphoid site NK cells expressing lower levels of effector molecules granzyme B, TNFα, Prf, Ifnγ and displaying reduced degranulation compared with their counterparts from blood, BM and spleen. For an in-depth analysis of tissue-mediated effects on NK cell subset functionality, we performed whole-transcriptome profiling on immature and mature NK cells isolated from blood, BM, spleen, lung and LN. Our analysis identified several effector molecules and NK cell surface receptors being differentially expressed between immature and mature NK cells. Furthermore, while mature NK cells of blood and tissues have similar transcriptional profiles, the transcriptional profiles of immature blood and tissue NK cells show tissue driven heterogeneity with differential expression of transcription factors, metabolic enzymes and NK cell surface receptors. Interestingly, the transcriptional signature of immature NK cells is reminiscent of the transcriptional signature of tissue resident memory T-cells showing increased expression of CD103, CD49a, CXCR6. We validated the expression of these markers using multiparameter flow cytometry and found that subset of immature NK cells in mucosal sites (lung and intestine) do indeed express markers of tissue residence. Additionally, by applying trajectory projection algorithm on NK cells from tissue sites, we show that resident NK cells comprise a distinct population from immature NK cells. Our study has identified novel, previously unidentified diversity of tissue NK cells. Phenotypic and transcriptional profiling data provide evidence for putative resident NK cells being present in certain tissue sites. Blood NK cells differ from their counterparts in tissues; especially, immature NK cells in tissues may be specifically trained to function in the tissue environment. Finally, due to the uniqueness of phenotypic, functional and transcriptional features of tissue NK cells, they may be more suited to fight cancer in situ in tissues. Citation Format: Pranay Dogra, Takashi Senda, Peter Szabo, Dustin Carpenter, Marta Toth, Puspa Thapa, Mark Snyder, Michelle Miron, Brahma Kumar, Donna L. Farber. Human NK cell distribution memory and residence in tissue sites [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B153.

Visualization of Phytophthora palmivora Infection in Oil Palm Leaflets with Fluorescent Proteins and Cell Viability Markers.

Bud rot (BR) is the most devastating disease affecting oil palm (Elaeis guineensis) crops in Colombia. Its causal agent, Phytophthora palmivora, initiates the infection in immature oil palm leaflets producing necrotic lesions, followed by colonization of opportunistic necrotrophs, which increases disease damage. To improve the characterization of the disease, we transformed P. palmivora using Agrobacterium tumefaciens-mediated transformation (ATMT) to include the fluorescent proteins CFP-SKL (peroxisomal localization), eGFP and mRFP1 (cytoplasmic localization). The stability of some transformants was confirmed by Southern blot analysis and single zoospore cultures; additionally, virulence and in vitro growth were compared to the wild-type isolate to select transformants with the greatest resemblance to the WT isolate. GFP-tagged P. palmivora was useful to identify all of the infective structures that are commonly formed by hemibiotrophic oomycetes, including apoplastic colonization and haustorium formation. Finally, we detected cell death responses associated with immature oil palm tissues that showed reduced susceptibility to P. palmivora infection, indicating that these tissues could exhibit age-related resistance. The aim of this research is to improve the characterization of the initial disease stages and generate cell biology tools that may be useful for developing methodologies for early identification of oil palm materials resistant or susceptible to BR.

Sacrococcygeal teratoma in children: Single center experience

Introduction: Sacrococcygeal teratoma (SCT) represent the most common benign and malignant germ cell tumors (GCT) in newborns and infants developing from primordial germ cells of the human embryo. Most of them are benign, however those presenting later in life and having major intra-pelvic component have greater malignant potential. Early and complete excision of the SCT has been the mainstay of successful management. The aim of this study is to describe the patient's details, management done, histopathology of the tumor, recurrence and the over all outcome of patients with SCT. Materials and Methods: Patients with histological diagnosis of sacrococcygeal teratoma during the period of January 2014 and October 2016 were included in this retrospective observational study. Data was collected regarding age, gender, mode of presentation, associated anomalies, Altman’s classification, investigations, treatment modality, tumor histology according to type of tumour with margin of resection, and outcome of management. All the diagnosed cases of SCT presenting during the above duration were included for study. Results: Twenty eight patients were included in the study. Out of them 75% were females and 25% were males. 32.14% presented during neonatal period. 78.57% presented with external mass. The tumour was completely resected by sacral approach in 22 patients and by combined abdominosacral approach in 06 patients. Recurrence was detected in one patients (3.57%); with Altman types 1 mature teratoma. Patients were followed for a duration ranged between 1 months and 3 years. Conclusion: Early diagnosis and early complete enblock excision of the tumor with the coccyx was mainstay of treatment with good prognosis. Late presentation and the presence of malignant changes are associated with poor prognosis. Intraoperative spillage of the tumor should be avoided. Overall survival of SCT is high. Keywords: Sacrococcygeal tumors, Germ cell tumor, Immature tissue.

Effects of Estrogen-Deficient State on Rotator Cuff Healing

Background: Rotator cuff retears after surgical repair are a concern, despite advances in operative techniques, but few studies have investigated the effects of the estrogen-deficient state on tendon-to-bone healing at the repair site. Purpose: We evaluated the effect of the estrogen-deficient state on tendon-to-bone healing after rotator cuff repair in an ovariectomized rat model. Study Design: Controlled laboratory study. Methods: Female Sprague Dawley rats underwent detachment and immediate repair of the supraspinatus tendon. Surgery was performed in 24 rats at 4.5 weeks of age 17 weeks after ovariectomy (OVX group) and in 24 age-matched control rats without ovariectomy (control group). Animals were sacrificed at 2, 4, 8, and 12 weeks after surgery for biomechanical and histological evaluations of reattachment. Bone mineral density (BMD) at the insertion site and cancellous bone in the humeral head was assessed by micro–computed tomography. Results: BMD was significantly lower both at the insertion site and in cancellous area in the OVX group than in the control group at weeks 2 to 12. Ultimate load to failure, ultimate stress, linear stiffness, and the Young modulus were significantly lower in the OVX group than in the control group at 2 and 4 weeks, but the difference was no longer significant at 8 and 12 weeks. At 2 and 4 weeks, relatively immature granulation tissue was observed in the OVX group compared with the control group. At 8 and 12 weeks after surgery, there were differences in the tendon-bone interface in the 2 groups: Direct insertion with well-established chondroid tissue was seen in the control group, and indirect insertion without chondroid tissue was seen in the OVX group. Consistently, the amount of chondroid tissue was greater and collagen organization was better in the control group than in the OVX group. Cells expressing cathepsin K were significantly more numerous both at the insertion site and in cancellous bone in the OVX group than in the control group. Conclusion: The estrogen-deficient state by ovariectomy, compared with control rats, led to decreased biomechanical properties and poor development of chondroid tissue that influenced the repair of the tendon insertion after surgery. Clinical Relevance: Agents that modulate bone metabolism might improve tendon-to-bone healing in patients with an estrogen-deficient state, such as postmenopausal women who undergo rotator cuff surgery.

Setting Up a Cryopreservation Programme for Immature Testicular Tissue: Lessons Learned After More Than 15 Years of Experience.

Young boys undergoing gonadotoxic treatments are at high risk of spermatogonial stem cell (SSC) loss and fertility problems later in life. Stem cell loss can also occur in specific genetic conditions, eg, Klinefelter syndrome (KS). Before puberty, these boys do not yet produce sperm. Hence, they cannot benefit from sperm banking. An emerging alternative is the freezing of testicular tissue aiming to preserve the SSCs for eventual autologous transplantation or in vitro maturation at adult age. Many fertility preservation programmes include cryopreservation of immature testicular tissue, although the restoration procedures are still under development. Until the end of 2018, the Universitair Ziekenhuis Brussel has frozen testicular tissues of 112 patients between 8 months and 18 years of age. Testicular tissue was removed in view of gonadotoxic cancer treatment (35%), gonadotoxic conditioning therapy for bone marrow transplantation (35%) or in boys diagnosed with KS (30%). So far, none of these boys had their testicular tissue transplanted back. This article summarizes our experience with cryopreservation of immature testicular tissue over the past 16 years (2002-2018) and describes the key issues for setting up a cryopreservation programme for immature testicular tissue as a means to safeguard the future fertility of boys at high risk of SSC loss.

Temporomandibular arthropathies: A retrospective study with histopathological characteristics.

BackgroundTo investigate the incidence of temporomandibular arthropathies diagnosed in a university center and to describe their histopathological characteristics.Material and MethodsTemporomandibular arthropathy cases with corresponding slides were selected from an oral and maxillofacial surgical pathology service. Cases of exclusively articular disc disease were not included.ResultsThe mean age was 31.3 years with a predominance of females (69.7%). Of these diagnoses, 53.6% were unilateral condylar hyperplasia, 17.8% were bony ankylosis, 14.3% were degenerative joint disease, 10.7% were osteochondroma, and 3.6% were synovial chondromatosis. Condylar hyperplasia presented as thick fibrocartilage and cartilage nests in the cancellous bone. Bony ankylosis exhibited lamellar bone and nests of chondrocytes. Degenerative joint disease presented as an irregular layer of fibrocartilage with areas of clustered chondrocytes and calcified cartilage. Osteochondroma of the condyle exhibited hyaline cartilage and areas of new bone formation. Synovial chondromatosis presented as immature cartilaginous tissue and randomly arranged chondrocytes.ConclusionsThe pathological alterations verified in these arthropathies involved diseases that were predominantly proliferative, i.e., unilateral condylar hyperplasia, osteochondroma and synovial chondromatosis of the tumor or pseudotumor type and bony ankylosis associated with callus formation of the reparative type, and less frequent degenerative changes for which the disease is so named. Key words:Temporomandibular joint, pathology, ankylosis, pathology, arthritis, degenerative, osteochondroma, chondromatosis, synovial.

Ontogeny of effective mechanical advantage in eastern cottontail rabbits (Sylvilagus floridanus).

Juvenile animals must survive in the same environment as adults despite smaller sizes, immature musculoskeletal tissues, general ecological naïveté and other limits of performance. Developmental changes in muscle leverage could constitute one mechanism to promote increased performance in juveniles despite ontogenetic limitations. We tested this hypothesis using a holistic dataset on growth and locomotor development in wild eastern cottontail rabbits (Sylvilagus floridanus) to examine ontogenetic changes in hindlimb muscle effective mechanical advantage (EMA). EMA is a dimensionless index of muscle leverage, equal to the quotient of average muscle lever length and the load arm length of the ground reaction force (GRF), effectively representing the magnitude of output force arising from a given muscle force. We found that EMA at the hip and ankle joints, as well as overall hindlimb EMA, significantly declined across ontogeny in S. floridanus, whereas EMA at the knee joint remained unchanged. Ontogenetic decreases in EMA were due to isometric scaling of muscle lever arm lengths alongside positive ontogenetic allometry of GRF load arm lengths - which in turn was primarily related to positive allometry of hindlimb segment lengths. Greater EMA limits the estimated volume of hindlimb extensor muscle that has to be activated in young rabbits, likely mitigating the energetic cost of locomotion and saving metabolic resources for other physiological functions, such as growth and tissue differentiation. An additional examination of limb growth allometry across a diverse sample of mammalian taxa suggests that ontogenetic decreases in limb joint EMA may be a common mammalian trend.

Fibrous Dysplasia in Maxillary Bone: Case Report

Fibrous dysplasia is a benign lesion, congenital, recurrent character and etiology unknown, caused by a bone modeling disorder with constant alterations of the normal bone being replaced by immature fibrous tissue. The lesion may involve one or more bones being the maxilla the facial bone more affected. We report a case of polyostotic fibrous dysplasia in a male patient complaining of increased volume in the right genital region. In this case, Computed Tomography (CT) was the main radiographic examination to demonstrate the extension and radiodensity that the lesion assumes in the craniofacial bones, being therefore of fundamental importance in the surgical planning and in the longitudinal follow-up of the operated patient. The diagnosis was confirmed by anatopathology and the treatment of choice was osteoplasty. This article also aims to review the main clinical, radiological, histopathological, differential diagnosis and Fibrous dysplasia treatment.

The effectiveness of the reparative processes stimulation of the platelet-rich plasma in case of the abdominal wall endoprosthetics

Relevance. The number of ventral hernias recurrence after abdominal wall endoprosthetics with the lightweight endoprosthesis is 8-10%.&#x0D; The purpose of the research is to study the morphological changes of the connective tissue, surrounding a lightweight polypropylene endoprosthesis, at the early stages of implantation at the use of platelet-rich autoplasma.&#x0D; Materials and methods. The results of experimental research of the course of wound process in the abdominal wall tissues during the implantation of the lightweight polypropylene prosthesis on 50 Chinchilla rabbits divided into 2 groups of 25 animal units each have been analyzed. Subaponeurotic implantation of the prosthesis was carried out in the first group of animals. In the second group, the platelet-rich autoplasma was twice introduced after the implantation of the prosthesis during surgery and on the third day of the postoperative period at the rate of 0,5 ml plasma per 1 cm2 of the endoprosthesis. The animals were taken away from the experiment on the 3rd, 7th, 10th, 14th and 21st day. Morphometric and histological study of reactive changes of tissue surrounding the implanted endoprosthesis was carried out.&#x0D; Results and its discussion. In the animals of the second group, the inflammatory response to prosthesis implantation was 1,3 times lower on the third day, 1,2 times lower on the 7th day, and 1,1 times lower on the 10th day, than in the first group. Fibroblastic response, on the contrary, has increased into 1,2 times on the third day, into 1,14 during the 7th day and into 1,18 during the 14th day after the introduction of autoplasma. The thickness of the connective tissue capsule in the second group was 1,57 times higher than in the first group on the 21st day of the experiment. At this time, in the first group, the immature connective tissue capsule has been represented by the loose collagen fibers and fibroblastic cells without separation into the layers. In the second group, a dense connective tissue frame has been formed by the mature collagen fibers arranged compactly and in parallel to each other. The layer-by-layer structure of the capsule is well expressed: the inner layer consists of the fibroblastic cells, and the outer layer consists of the mature collagen fibers.&#x0D; Conclusion. The use of platelet-rich plasma to stimulate reparative processes in the implantation of the lightweight polypropylene prosthesis in the abdominal wall is pathogenetically justified and effective.

Expression analysis of genes associated with sucrose accumulation and its effect on source-sink relationship in high sucrose accumulating early maturing sugarcane variety.

Sucrose synthesis/accumulation in sugarcane depends on the source-sink communication wherein source responds to sink demand for photoassimilate supply. Sucrose in stalk (sink) acts as signal, and sends feedback to restrain further synthesis of sucrose by regulating photosynthetic efficiency of leaves (source). Hence sucrose synthesis/accumulation is controlled by many genes and regulatory sequences including 3 invertases (SAI, CWI, NI), sucrose synthase (SuSy) and sucrose phosphate synthase (SPS). SPS and invertase play key role in enhancing sink strength which ultimately promotes greater sucrose accumulation in the sink tissues. In present study, a significant positive correlation was found between sucrose% of source and sink tissues which was greater in the top (R 2 = 0.679) than middle (R 2 = 0.580) and bottom (R 2 = 0.518) internodes, depicting that sucrose accumulation in the stalk bears a direct relation with sucrose translocation efficiency from source. Results indicated an increased sucrose% with maturity, while reducing sugar content decreased with crop growth. qRT-PCR results exhibited an elevated expression of invertase in immature sink tissues depicting increased sink requirement, which declined with maturity. Similarly, increased PEP carboxylase gene expression as observed supported the fact that higher sink demand results in enhanced photosynthetic rate and thus influences the source activity. SPS was found active at initial stage of cane development indicating its role in sucrose synthesis. Thus by studying expression patterns of the different genes both, in source and sink tissues, a better understanding of the sucrose accumulation pathway in sugarcane is possible, which in turn can help in elucidating ways to enhance sucrose concentration in sink.

Screening Examination of Premature Infants for Retinopathy of Prematurity.

This policy statement revises a previous statement on screening of preterm infants for retinopathy of prematurity (ROP) that was published in 2013. ROP is a pathologic process that occurs in immature retinal tissue and can progress to a tractional retinal detachment, which may then result in visual loss or blindness. For more than 3 decades, treatment of severe ROP that markedly decreases the incidence of this poor visual outcome has been available. However, severe, treatment-requiring ROP must be diagnosed in a timely fashion to be treated effectively. The sequential nature of ROP requires that infants who are at-risk and preterm be examined at proper times and intervals to detect the changes of ROP before they become destructive. This statement presents the attributes of an effective program to detect and treat ROP, including the timing of initial and follow-up examinations.

Biopolymer gels as a basis of cryoprotective medium for testicular tissue of rats.

Cryopreservation of testis tissue is a promising approach to save fertility in prepubertal boys under going gonadotoxic cancer therapies. The using biopolymers as a basis of cryoprotective medium can be effective for the optimization of cryopreservation protocols of immature testicular tissue. The research purpose was to determine morphological parameters and metabolic activity of seminiferous tubules of immature rat testes under exposure to cryoprotective solution (DMSO) based on collagen or fibrin gels (CG or FG) as one of the first stages of developing the cryopreservation protocol. It was found that 30-min exposure of tissue samples to CG and FG with 0.6M DMSO did not impair the spermatogenic epithelium and metabolic activity of the cells (MTT test and total lactate dehydrogenase activity). The use of FG at the time of exposure of 45min did not lead to significant changes in the metabolic activity in contrast to other groups. The findings could be used to substantiate and develop the effective techniques for cryopreservation of immature seminiferous tubules.

Influence of temperature, serum, and gonadotropin supplementation in short- and long-term organotypic culture of human immature testicular tissue

To study how temperature, serum, and gonadotropin supplementation affect the organotypic culture of human immature testicular tissue (ITT) invitro. Experimental basic science study. Reproductive biology laboratory. ITT from 4 boys with cancer that had testicular tissue cryopreserved as part of their fertility preservation treatment. Invitro organotypic culture of ITT, exposed to different temperatures (37°C vs. 34°C), serum (fetal bovine serum [FBS] vs. Knockout Serum Replacement [KOS]), and gonadotropin supplementation (with and without FSH and LH). Characterization of the tissue was performed at days 0, 14, and 70 with the use of reverse-transcription quantitative polymerase chain reaction, terminal deoxynucleotide transferase-mediated dUTP nick-end labeling, histologic analysis by means of hematoxylin-eosin staining, and immunohistochemical staining. Hormonal secretion was determined at days 3, 14, 28, and 70by means of immunofluorescent assay. The 37°C conditions showed an accelerated loss of tubular morphology and higher intratubular apoptosis. KOS supplementation triggered the up-regulation of STAR, SOX9, DAZL, DDX4, PLZF, and UTF1, the percentage of SOX9+/androgen receptor (AR)-positive mature Sertoli cells at day 14, and testosterone secretion. Gonadotropin supplementation increased the numbers of both undifferentiated UTF1+ spermatogonia and premeiotic VASA+/SYCP3+ spermatogonia at day 14, and the number of SOX9+ Sertoli cells at day 70. The low SOX9+/AR+ colocalization, the disorganized pattern of ZO-1, and the progressive decrease of antimüllerian hormone secretion indicated inefficient Sertoli cell maturation invitro. The 34°C condition in KOS showed the best results for the survival of both spermatogonia and Sertoli cells. FSH/LH supplementation also improved long-term survival of Sertoli cells and the maturation of spermatogonia up to meiotic initiation in short-term culture.

Genetic transformation of einkorn (Triticum monococcum L. ssp. monococcum L.), a diploid cultivated wheat species

BackgroundDomesticated einkorn (Triticum monococcum L.) is one of the oldest cultivated cereal crops in the world. Its small genome size (~ 5.7 GB), low ploidy (2n = 2x = 14, AmAm) and high genetic polymorphism make this species very attractive for use as a diploid model for understanding the genomics and proteomics of Triticeae. Einkorn, however, is still a recalcitrant monocotyledonous species for the application of modern biotechnologies, including transgenesis. This paper reports the factors that may influence transgene delivery, integration, expression and inheritance in einkorn.ResultsIn this study, we report the successful genetic transformation of einkorn using biolistic-mediated DNA delivery. Immature embryo-derived tissues of spring einkorn were bombarded with a plasmid containing the reporter gene GFP (green fluorescent protein) driven by the rice actin promoter (act1) and the selectable bar gene (bialaphos resistance gene) driven by the maize ubiquitin promoter (ubi1). Adjustments to various parameters such as gas pressure, microcarrier size and developmental stage of target tissue were essential for successful transient and stable transformation. Bombarded einkorn tissues are recalcitrant to regenerating plants, but certain modifications of the culture medium have been shown to increase the production of transgenic events. In various experiments, independent transgenic plants were produced at frequencies ranging from 0.0 to 0.6%. Molecular analysis, marker gene expression and herbicide treatment demonstrated that gfp/bar genes were stably integrated into the einkorn genome and successfully inherited over several generations. The transgenes, as dominant loci, segregated in both Mendelian and non-Mendelian fashion due to multiple insertions. Fertile homozygous T1-T2 populations of transgenic einkorn that are resistant to herbicides were selected.ConclusionTo the best of our knowledge, this is the first report of the production of genetically modified einkorn plants. We believe that the results of our research could be a starting point for the application of the current biotechnological-based technologies, such as transgenesis and genome editing, to accelerate comparative functional genomics in einkorn and other cereals.

Haploid Germ Cells Generated in Organotypic Culture of Testicular Tissue From Prepubertal Boys.

While in mice various studies have described the completion of spermatogenesis in vitro using either organotypic culture of prepubertal testicular tissue or 3D culture of isolated cells, in humans it has not been possible to achieve germ cell differentiation from immature testicular tissue (ITT). In our study, we evaluated the ability of human ITT to differentiate via a long-term organotypic culture of frozen–thawed 1 mm3 testicular fragments from five prepubertal boys in two different culture media. Tissue and supernatants were analyzed at regular intervals up to day 139. Sertoli cell (SC) viability and maturation was evaluated using immunohistochemistry (IHC) for SOX9, GDNF, anti-Mullerian hormone (AMH) and androgen receptor (AR), and AMH concentration in supernatants. Spermatogonia (SG) and proliferating cells were identified by MAGE-A4 (for SG) and Ki67 (for proliferating cells) via immunohistochemistry (IHC). Apoptotic cells were studied by active caspase 3. To evaluate Leydig cell (LC) functionality testosterone was measured in the supernatants and steroidogenic acute regulatory protein (STAR) IHC was performed. Germ cell differentiation was evaluated on Hematoxylin-Eosin histological sections, via IHC for synaptonemal complex 3 (SYCP3) for spermatocytes, Protein boule-like (BOLL) for spermatocytes and round spermatids, angiotensin-converting enzyme (ACE), protamine 2 and transition protein 1 (for elongated spermatids) and via chromogenic in situ hybridization (CISH). We reported the generation of meiotic and postmeiotic cells after 16 days of culture, as shown by the histological analyses, the presence of differentiation markers and the increase of haploid germ cells. We showed SC viability and maturation by a decrease of AMH secretion in the supernatants (p ≤ 0.001) while the number of SOX9 positive cells did not show any variation. A decrease of spermatogonia (p ≤ 0.001) was observed. The number of apoptotic cells did not vary. LC functionality was shown by the increase in STAR expression (p ≤ 0.007) and a peak in testosterone secretion, followed by a reduction (p ≤ 0.001) with stabilization. According to our knowledge, this is the first report of generation of haploid cells in human ITT. Differentiating germ cells have to be further evaluated for their ability to complete differentiation, their fecundability and epigenetic characteristics.